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Extracting Cellular Organelles

Issuing time:2018-02-15 05:37

Due to the differences in size, homogenizers are sometimes capable of lysing cells while preserving the organelles contained within.

Tips for Isolating Intact Organelles from Cells

As with any application where something needs to be left intact, the key is doing just enough to get the job done and no more. The point between cell lysis and organelle lysis is narrow, and the smaller the ratio of cell size / organelle size the narrower that window is. It may take a fair amount of testing to optimize for yield of intact organelles. We recommend using spare cells or tissue such that you do can optimize your protocol prior to using valuable experimental samples.

When testing to determine a good protocol, start with a low speed setting then gradually increase the speed if the settings fail to lyse the cells or dissociate the tissue. You will almost always achieve better organelle viability if you use a longer, slower run than a shorter, faster run.

Be sure to process your cells or tissue in a medium that is appropriate for the recovery of viable organelles. Do not use detergents or other chemicals to aid in cell lysis unless you know that your specific organelles are resistant to the substance.

Choosing a Homogenizer for Organelle Isolation

Numerous homogenization technologies may be used for organelle isolation.

If starting from cell culture, a high-pressure homogenizer may be used. This method is particularly useful for larger volumes of sample. Ideally you will want a homogenizer that does not make use of a plate or blade at which the high-pressure homogenate stream is directed. Regardless, the trick will be to find the pressure setting which causes just enough shear to lyse the comparatively larger cells while leaving intact the smaller organelles.

Bead mills are good options for smaller volumes, although optimization can be tricky. Cells require small beads for efficient lysis, but to leave organelles intact you want to avoid using beads which are too small. It may be worth trying slightly larger beads than you might ordinarily consider for cell lysis, or simply reducing the quantity of beads somewhat.

Rotor-stator homogenizers can be used as well. If using a rotor-stator, ensure that you are using a probe which is large enough to efficiently mix the sample - you do not want some areas of the sample to be processed repeatedly while others are missed.

Mortars and pestles are difficult to optimize due to reproducibility issues and are therefore not recommended. Never use ultrasonics for organelle extraction, as they will lyse organelles in a highly efficient manner.

Whichever type of homogenizer you choose, it is best to select an instrument with a very low minimum power or speed setting such that you can process your samples as gently as may be required.


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